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Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a public health threat for which preventive and therapeutic agents are urgently needed. Neutralizing antibodies are a key class of therapeutics that may bridge widespread vaccination campaigns and offer a treatment solution in populations less responsive to vaccination. Here, we report that high-throughput microfluidic screening of antigen-specific B cells led to the identification of LY-CoV555 (also known as bamlanivimab), a potent anti-spike neutralizing antibody from a hospitalized, convalescent patient with coronavirus disease 2019 (COVID-19). Biochemical, structural, and functional characterization of LY-CoV555 revealed high-affinity binding to the receptor-binding domain, angiotensin-converting enzyme 2 binding inhibition, and potent neutralizing activity. A pharmacokinetic study of LY-CoV555 conducted in cynomolgus monkeys demonstrated a mean half-life of 13 days and a clearance of 0.22 ml hour-1 kg-1, consistent with a typical human therapeutic antibody. In a rhesus macaque challenge model, prophylactic doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract in samples collected through study day 6 after viral inoculation. This antibody has entered clinical testing and is being evaluated across a spectrum of COVID-19 indications, including prevention and treatment.

LY-CoV555, a SARS-CoV-2 spike protein–specific antibody, neutralizes SARS-CoV-2 and protects the airways of nonhuman primates against infection., A monoclonal for SARS-CoV-2 Among the most promising therapeutic options for individuals with coronavirus disease 2019 (COVID-19) are monoclonal antibodies (mAbs). In this study, Jones et al. identified, characterized, and tested one such mAb, LY-CoV555, in vitro and in vivo. They found that LY-CoV555 bound to the severe acute respiratory distress syndrome coronavirus-2 (SARS-CoV-2) spike protein and prevented its interaction with angiotensin-converting enzyme 2. Prophylactic treatment with LY-CoV555 protected the upper and lower respiratory tracts of nonhuman primates from becoming infected with SARS-CoV-2. Together, these data support the clinical use of LY-CoV555 for treating patients with COVID-19., Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a public health threat for which preventive and therapeutic agents are urgently needed. Neutralizing antibodies are a key class of therapeutics that may bridge widespread vaccination campaigns and offer a treatment solution in populations less responsive to vaccination. Here, we report that high-throughput microfluidic screening of antigen-specific B cells led to the identification of LY-CoV555 (also known as bamlanivimab), a potent anti-spike neutralizing antibody from a hospitalized, convalescent patient with coronavirus disease 2019 (COVID-19). Biochemical, structural, and functional characterization of LY-CoV555 revealed high-affinity binding to the receptor-binding domain, angiotensin-converting enzyme 2 binding inhibition, and potent neutralizing activity. A pharmacokinetic study of LY-CoV555 conducted in cynomolgus monkeys demonstrated a mean half-life of 13 days and a clearance of 0.22 ml hour−1 kg−1, consistent with a typical human therapeutic antibody. In a rhesus macaque challenge model, prophylactic doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract in samples collected through study day 6 after viral inoculation. This antibody has entered clinical testing and is being evaluated across a spectrum of COVID-19 indications, including prevention and treatment.

We describe a bispecific dual-antagonist antibody against human B cell activating factor (BAFF) and interleukin 17A (IL-17). An anti-IL-17 single-chain variable fragment (scFv) derived from ixekizumab (Taltz®) was fused via a glycine-rich linker to anti-BAFF tabalumab. The IgG-scFv bound both BAFF and IL-17 simultaneously with identical stoichiometry as the parental mAbs. Stability studies of the initial IgG-scFv revealed chemical degradation and aggregation not observed in either parental antibody. The anti-IL-17 scFv showed a high melting temperature (Tm) by differential scanning calorimetry (73.1°C), but also concentration-dependent, initially reversible, protein self-association. To engineer scFv stability, three parallel approaches were taken: labile complementary-determining region (CDR) residues were replaced by stable, affinity-neutral amino acids, CDR charge distribution was balanced, and a H44-L100 interface disulfide bond was introduced. The Tm of the disulfide-stabilized scFv was largely unperturbed, yet it remained monodispersed at high protein concentration. Fluorescent dye binding titrations indicated reduced solvent exposure of hydrophobic residues and decreased proteolytic susceptibility was observed, both indicative of enhanced conformational stability. Superimposition of the H44-L100 scFv (PDB id: 6NOU) and ixekizumab antigen-binding fragment (PDB id: 6NOV) crystal structures revealed nearly identical orientation of the frameworks and CDR loops. The stabilized bispecific molecule LY3090106 (tibulizumab) potently antagonized both BAFF and IL-17 in cell-based and in vivo mouse models. In cynomolgus monkey, it suppressed B cell development and survival and remained functionally intact in circulation, with a prolonged half-life. In summary, we engineered a potent bispecific antibody targeting two key cytokines involved in human autoimmunity amenable to clinical development.

Altered coagulation and inflammation contribute to the pathogenesis of ischemic renal injury. Thrombomodulin is a necessary factor in the anticoagulant protein C pathway and has inherent anti-inflammatory properties. We studied the effect of soluble thrombomodulin (sTM) in a hypoperfusion model of ischemic kidney injury. To markedly reduce infrarenal aortic blood flow and femoral arterial pressures, we clamped the suprarenal aorta of rats, occluding them 90%, for 60 min. Reversible acute kidney injury (AKI) occurred at 24 h in rats subjected to hypoperfusion. Histologic analysis at 24 h revealed acute tubular necrosis (ATN), and intravital two-photon microscopy showed flow abnormalities in the microvasculature and defects of endothelial permeability. Pretreatment with rat sTM markedly reduced both I-R-induced renal dysfunction and tubular histologic injury scores. sTM also significantly improved microvascular erythrocyte flow rates, reduced microvascular endothelial leukocyte rolling and attachment, and minimized endothelial permeability to infused fluorescence dextrans, assessed by intravital quantitative multiphoton microscopy. Furthermore, sTM administered 2 h after reperfusion protected against ischemia-induced renal dysfunction at 24 h and improved survival. By using an sTM variant, we also determined that the protective effects of sTM were independent of its ability to generate activated protein C. These data suggest that sTM may have therapeutic potential for ischemic AKI.

Administration of activated protein C (APC) protects from renal dysfunction, but the underlying mechanism is unknown. APC exerts both antithrombotic and cytoprotective properties, the latter via modulation of protease-activated receptor-1 (PAR-1) signaling. We generated APC variants to study the relative importance of the two functions of APC in a model of LPS-induced renal microvascular dysfunction. Compared with wild-type APC, the K193E variant exhibited impaired anticoagulant activity but retained the ability to mediate PAR-1-dependent signaling. In contrast, the L8W variant retained anticoagulant activity but lost its ability to modulate PAR-1. By administering wild-type APC or these mutants in a rat model of LPS-induced injury, we found that the PAR-1 agonism, but not the anticoagulant function of APC, reversed LPS-induced systemic hypotension. In contrast, both functions of APC played a role in reversing LPS-induced decreases in renal blood flow and volume, although the effects on PAR-1-dependent signaling were more potent. Regarding potential mechanisms for these findings, APC-mediated PAR-1 agonism suppressed LPS-induced increases in the vasoactive peptide adrenomedullin and infiltration of iNOS-positive leukocytes into renal tissue. However, the anticoagulant function of APC was responsible for suppressing LPS-induced stimulation of the proinflammatory mediators ACE-1, IL-6, and IL-18, perhaps accounting for its ability to modulate renal hemodynamics. Both variants reduced active caspase-3 and abrogated LPS-induced renal dysfunction and pathology. We conclude that although PAR-1 agonism is solely responsible for APC-mediated improvement in systemic hemodynamics, both functions of APC play distinct roles in attenuating the response to injury in the kidney.

Skeletal muscle wasting occurs in a great majority of cancer patients with advanced disease and is associated with a poor prognosis and decreased survival. Myostatin functions as a negative regulator of skeletal muscle mass and has recently become a therapeutic target for reducing the loss of skeletal muscle and strength associated with clinical myopathies. We generated neutralizing antibodies to myostatin to test their potential use as therapeutic agents to attenuate the skeletal muscle wasting due to cancer. We show that our neutralizing antimyostatin antibodies significantly increase body weight, skeletal muscle mass, and strength in non–tumor-bearing mice with a concomitant increase in mean myofiber area. The administration of these neutralizing antibodies in two preclinical models of cancer-induced muscle wasting (C26 colon adenocarcinoma and PC3 prostate carcinoma) resulted in a significant attenuation of the loss of muscle mass and strength with no effect on tumor growth. We also show that the skeletal muscle mass– and strength-preserving effect of the antibodies is not affected by the coadministration of gemcitabine, a common chemotherapeutic agent, in both non–tumor-bearing mice and mice bearing C26 tumors. In addition, we show that myostatin neutralization with these antibodies results in the preservation of skeletal muscle mass following reduced caloric intake, a common comorbidity associated with advanced cancer. Our findings support the use of neutralizing antimyostatin antibodies as potential therapeutics for cancer-induced muscle wasting. Mol Cancer Ther; 14(7); 1661–70. ©2015 AACR.

Human follistatin is a regulatory glycoprotein with widespread biological functions including anti-inflammatory activities, wound healing properties and muscle stimulating effects. The role of follistatin in a wide range of biological activities shows promise for potential clinical application, which has prompted considerable interest in the investigation of the protein as a potential disease modifying agent. In spite of this potential, the development of follistatin as a broad use biotherapeutic has been severely hindered by a poor understanding and characterization of its pharmacokinetic and pharmacodynamic (PK/PD) relationships. Therefore, to better define these relationships, we performed in-depth analyses of the PK/PD relationships of native follistatin-315. Our data indicates that the intrinsic PK/PD properties of native follistatin-315 are poorly suited for acting as a parentally administered biotherapeutic with broad systemic effects. Herein, we leveraged protein engineering to modify the PK characteristics of the native molecule by fusing follistatin-315 to a murine IgG1 Fc and removing the intrinsic heparan sulfate binding activity of follistatin. The engineered variant molecule had ~100- and ~1600-fold improvements in terminal half-life and exposure, respectively. In contrast to the native follistatin-315, the variant showed a robust, dose-dependent pharmacological effect when administered subcutaneously on a weekly basis in mouse models of muscle atrophy and degeneration. These studies highlight the underappreciated and critical relationship between optimizing multiple physical and chemical properties of follistatin on its overall PK/PD profile. Moreover, our findings provide the first documented strategy towards the development of a follistatin therapeutic with potential utility in patients affected with skeletal muscle diseases.